The exact mutagenic consequences of introduction of defined lesions into DNA are being examined in a forward mutational system using M13mp2 DNA and capable of detecting a wide spectrum of mutational events, both at and some distance away from the actual site of damage. The system has first been applied to depurination the loss of a purine base from DNA. This is a frequent spontaneous lesion as well as a common intermediate in the repair of many other types of DNA damage. Depurination is highly mutagenic, as determined by transfection of depurinated M13mp2 DNA into SOS-induced compentent E. coli cells. DNA sequence analysis of 211 mutants demonstrates that most mutations are base substitutions reflecting insertion of dAMP opposite the non-coding abasic site and resulting in characteristic transversions. In order to determine the effects of a single abasic site placed at a defined position in the DNA, a general approach has been developed which should be applicable to many diverse types of premutagenic lesions. This procedure utilizes uracil containing DNA templates for standard in vitro reactions to incorporate the lesion into a covalently closed circular, and thus biologically active, molecule. The product of this reaction is then treated to yield, as the sole source of biological activity, complementary strand circles containing to lesion of interest at a single site and at essentially 100% frequency. In addition to its utility in studying lesions in DNA, the technique can be applied to standard site specific mutagenesis protocols to obtain very high efficiency even without selection.